Hydrolysis of inositol phosphates by plant cell extracts.

A gel-filtered soluble fraction prepared from suspension-cultured Nicotiana tabacum cells hydrolysed inositol mono-, bis- and tris-phosphates. At a concentration of 7.5 microM the rates of hydrolysis followed the sequence Ins(1,4,5)P3 greater than Ins(1,4)P2 greater than Ins(4)P congruent to Ins(1)P. The major products of Ins(1,4,5)P3 hydrolysis identified by h.p.l.c. were Ins(1,4)P2 and Ins(4,5)P2. Ins(1,4)P2 was hydrolysed exclusively to Ins(4)P. The inclusion of Ca2+ in the incubation buffer markedly stimulated the hydrolysis of all the inositol phosphate substrates. Under identical conditions, Ca2+ inhibited the hydrolysis of inositol phosphates by soluble extracts prepared from rat brain. Half-maximal stimulation of Ins(1,4)P2 hydrolysis was obtained at free [Ca2+] of 0.6 and 1.2 microM when the Mg2+ concentration in the incubations was 0.3 and 1.0 mM respectively. This effect of Ca2+ was exerted solely by increasing the Vmax. of hydrolysis without affecting the Km for Ins(1,4)P2. Again, in contrast with brain, the hydrolysis of inositol bis- or mono-phosphates was insensitive to high concentrations of Li+. We conclude that plants contain specific Li+-insensitive inositol phosphate phosphatases that are regulated by low concentrations of Ca2+ in a manner which is different from that observed in mammalian tissues.     

Angiotensin II-induced increase in inositol 1,4,5-trisphosphate in cultured rat mesangial cells: evidence by refined high performance liquid chromatography

Angiotensin II-induced change in inositol phosphates were studied in cultured rat mesangial cells prelabeled with [3H]myo-inositol. By using anion-exchange high performance liquid chromatography, we could analyzed the change in inositol mono-, bis-, and tris-phosphate more rapidly and easily with higher resolution than the previously reported methods. Angiotensin II rapidly increased inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate within 15 sec, followed by an increase in inositol 1-monophosphate at 30 sec. Angiotensin II-induced increases in inositol phosphates were dose-dependent and completely blocked by saralasin. These results indicate that angiotensin II induces the production of inositol phosphates including inositol 1,4,5-trisphosphate, an intracellular Ca2+-releasing factor, in cultured rat mesangial cells.     

Dopamine Receptor Stimulation Does Not Affect Phosphoinositide Hydrolysis in Slices of Rat Striatum

The effect of dopamine receptor stimulation on the accumulation of labelled inositol phosphates in rat striatal slices under basal and stimulated conditions was examined following preincubation with [3H]inositol. Incubation of striatal slices with the selective D-1 agonist SKF 38393 or the selective D-2 agonist LY 171555 for 5 or 30 min did not affect the basal accumulation of labelled inositol mono-, bis-, tris-, and tetrakisphosphate. Resolution by HPLC of inositol trisphosphate into inositol-1,3,4-tris-phosphate and inositol-1,4,5-trisphosphate isomers revealed that under basal conditions dopamine did not influence the accumulation of inositol-1,4,5-trisphosphate. Depolarisation evoked by KCl, or addition of the muscarinic receptor agonist carbachol, produced a marked increase in the accumulation of labelled inositol phosphates in both the presence and absence of lithium. Addition of dopamine did not reduce the ability of KCl or carbachol to increase inositol phospholipid hydrolysis. In the presence of lithium, dopamine (100 microM) enhanced KCl-stimulated inositol phospholipid hydrolysis, but this effect appears to be mediated by alpha 1 adrenoceptors because it was blocked by prazosin. SKF 38393 (10 microM) or LY 171555 (10 microM) also did not affect carbachol-stimulated inositol phospholipid hydrolysis. These data, in contrast to recent reports, suggest that striatal dopamine receptors do not appear to be linked to inositol phospholipid hydrolysis.     

Regional Differences in the Coupling of Muscarinic Receptors to Inositol Phospholipid Hydrolysis in Guinea Pig Brain

The differential effects of muscarinic agents on inositol phospholipid hydrolysis and the role in this process of putative muscarinic receptor subtypes (M1 and M2) were investigated in three regions of guinea pig brain. Addition of the agonist oxotremorine-M to slices of neostriatum, cerebral cortex, or hippocampus incubated in the presence of myo-[2-3H]inositol and Li+ resulted in a large accumulation of labeled inositol phosphates (733, 376, and 330% of control, respectively). In each tissue, the principal product formed was myo-inositol 1-phosphate (59-86%), with smaller amounts of glycerophosphoinositol and inositol bisphosphate. Only trace amounts of inositol trisphosphate could be detected. Regional differences were observed in the capacity of certain partial agonists to evoke inositol lipid hydrolysis, the most notable being that of bethanechol, which was four times more effective in the neostriatum than in either the cerebral cortex or hippocampus. In addition, the full agonists, oxotremorine-M and carbamoylcholine, were more potent stimulators of inositol phosphate release in the neostriatum than in the cerebral cortex. The putative M1 selective agonist 4-m-chlorophenylcarbamoyloxy-2-butynyl trimethyl ammonium chloride had little stimulatory effect in any brain region, whereas the putative M1 selective antagonist pirenzepine blocked the enhanced release of inositol phosphates with high affinity in the cerebral cortex and hippocampus (Ki = 12.1 and 13.9 nM; "M1") but with a lower affinity in the neostriatum (Ki = 160 nM; "M2"). In contrast to its differential effects on stimulated inositol lipid hydrolysis, no regional differences were observed in the capacity of pirenzepine to displace [3H]quinuclidinyl benzilate, a muscarinic antagonist, bound to membrane fractions. Atropine, an antagonist that does not discriminate between receptor subtypes, inhibited the enhanced release of inositol phosphates with similar affinities in the three regions (Ki = 0.40-0.60 nM). The results indicate that by measurement of inositol lipid hydrolysis, regional differences in muscarinic receptor coupling characteristics become evident. These differences, which are not readily detected by radioligand binding techniques, might be accounted for by either the presence of functionally distinct receptor subtypes, or alternatively, by regional variations in the efficiency of muscarinic receptor coupling to inositol lipid hydrolysis.     

Autohydrolysis of phytic acid.

The autohydrolysis of phytic acid at 120 degrees C resulted in the formation of most of the phosphate esters of myo-inositol in varying amounts depending upon the reaction time. Eighteen of the 39 chromatographically distinct myo-inositol mono-, bis-, tris-, tetrakis-, pentakis-, and hexakisphosphates have been characterized using two different HPLC systems. These myo-inositol phosphates were partially purified by preparative anion-exchange chromatography under acidic and alkaline elution conditions. The combination of these two methods provides a two-tiered chromatographic approach to the rapid and sensitive identification of inositol phosphates in complex mixtures. Identification of the products was confirmed by 1D and 2D (1)H NMR analysis. The analytical procedure was applied to the autohydrolysis of the mixture of inositol phosphates from corn steep water.     

Stimulation of phosphoinositide hydrolysis by oxytocin and the mechanism by which oxytocin controls prostaglandin synthesis in the ovine endometrium.

Slices of caruncular endometrium from steroid-treated ovariectomized sheep were incubated with myo-[2-3H]inositol to label tissue phosphatidylinositol. Effects of oxytocin were determined on the rate of incorporation of radioactivity into phosphatidylinositol and on the hydrolysis of phosphoinositides to inositol phosphates and diacylglycerol. Incorporation of radioactivity into phosphatidylinositol was linear during 2 h incubations; 10(-7) M (100 nM)-oxytocin caused a 2.8-fold increase in the rate of incorporation. In the presence of Li+, addition of 10(-7) M-oxytocin to slices in which phosphatidylinositol was pre-labelled caused mean increase of 40-fold in the incorporation of radioactivity into inositol mono-, bis- and tris-phosphates. Inositol 1,3,4-trisphosphate was quantitatively the major trisphosphate formed. The action of oxytocin on phosphoinositide hydrolysis was dose- and time-dependent, occurring at concentrations within the range observed in plasma during episodes of secretion in vivo, and with a time course comparable with that of the action of oxytocin on uterine prostaglandin production. The effect of oxytocin on incorporation of radioactivity into inositol phosphates was not affected by inhibitors of prostaglandin synthesis. Diacylglycerol 1- and 2-lipases in caruncular endometrium converted up to 72% of added 2-[3H]arachidonyldiacylglycerol into [3H]arachidonic acid during 30 min incubations at pH 7.0. Caruncular endometrium contained 1.49 mumol of phosphatidylinositol/g, representing approx. 0.2 mumol/g of phosphatidylinositol arachidonic acid. It is proposed that the stimulation of endometrial prostaglandin synthesis by oxytocin is accounted for by increased hydrolysis of phosphoinositides to diacylglycerol and inositol phosphates with subsequent release of arachidonic acid from diacylglycerol.     

Hydrolysis of inositol phospholipids induced by stimulation of the T cell antigen receptor complex in antigen-specific, murine helper T cell clones. Requirement for exogenous calcium

Two murine, keyhole limpet hemocyanin-specific, Th cell clones were studied for their ability to respond to antibody-mediated stimulation of the TCR complex or to Ag-pulsed accessory cells by hydrolyzing inositol phospholipids. Both clones were positive for the determinant expressed on the epsilon chain of CD3 that is recognized by the mAb, 145-2C11 (2C11 mAb); one clone also expressed the V beta 8 epitope of the alpha/beta chains of the TCR recognized by the F23.1 mAb. Treatment of these cells with 2C11 or F23.1 mAb adsorbed onto polystyrene beads induced a time-dependent accumulation of inositol phosphates (IP). Keyhole limpet hemocyanin-pulsed accessory cells which expressed the appropriate MHC phenotype also induced IP accumulation, whereas no response was induced by medium-treated or MHC congenic accessory cells. The hydrolysis of inositol phospholipids induced by TCR perturbation depended upon the presence of exogenous Ca2+; Mg2+ did not substitute for Ca2+. Treatment of cells with ionomycin at concentrations up to 30 microM was unable to induce hydrolysis of inositol phospholipids, indicating that entrance of Ca2+ was itself insufficient to generate IP. Stimulated IP generation was rapidly blocked upon addition of EGTA to the incubation medium. Reducing the level of exogenous Ca2+ decreased the production of inositol mono-, bis-, and trisphosphate isomers similarly, suggesting that extracellular Ca2+ was required for the initiation of the hydrolysis rather than affecting phospholipase C affinity for its substrates. We concluded that activation of inositol phospholipid hydrolysis by perturbation of the TCR complex in the Th cell clones under investigation displays a Ca2+-dependent component which is likely to be proximal to IP generation.     

Stimulatory effects of atrial natriuretic factor on phosphoinositide hydrolysis in cultured bovine aortic smooth muscle cells

The effects of atrial natriuretic factor (ANF) on phosphoinositide hydrolysis were examined in preparations of cultured bovine aortic smooth muscle cells. In homogenates or particulate fractions from cultured bovine aortic smooth muscle cells, ANF and atriopeptin I increased the formation of inositol phosphates and GTPase activity. The effects on inositol phosphates were markedly enhanced with guanosine 5'[gamma-thio]triphosphate. Both atrial peptides also stimulated the formation of diacylglycerol in intact cultured cells. In these experiments, atriopeptin I was about 10-fold more potent than ANF. These studies indicate that atrial peptides have stimulatory effects on phosphoinositide hydrolysis which are mediated through a guanine nucleotide regulatory protein. The greater potency of atriopeptin I on GTPase activity and the accumulation of inositol phosphates suggests that the nonguanylate cyclase-coupled receptor for ANF (ANF-R2) mediates the stimulatory effects of ANF on phosphoinositide hydrolysis through a guanine nucleotide regulatory protein.     

Inositol 1,4,5-triphosphate phosphatase activity in membranes isolated from amphibian skeletal muscle [corrected

The hydrolysis of [3H]inositol 1,4,5-trisphosphate by a soluble fraction and by isolated transverse tubule and sarcoplasmic reticulum membranes from frog skeletal muscle was studied. Transverse tubule membranes displayed rates of hydrolysis several-fold higher than those of sacroplasmic reticulum and soluble fraction; Km and Vmax were 25.2 microM and 44.1 nmol/mg/min, respectively. Transverse tubule membranes sequentially hydrolyzed inositol trisphosphate to inositol bisphosphate, inositol 1-phosphate and inositol, indicating that these membranes have inositol bis- and monophosphatases in addition to inositol trisphosphatase.     

Changes in the levels of inositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides.

The formation of inositol phosphates in response to agonists was studied in brain slices, parotid gland fragments and in the insect salivary gland. The tissues were first incubated with [3H]inositol, which was incorporated into the phosphoinositides. All the tissues were found to contain glycerophosphoinositol, inositol 1-phosphate, inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate, which were identified by using anion-exchange and high-resolution anion-exchange chromatography, high-voltage paper ionophoresis and paper chromatography. There was no evidence for the existence of inositol 1:2-cyclic phosphate. A simple anion-exchange chromatographic method was developed for separating these inositol phosphates for quantitative analysis. Stimulation caused no change in the levels of glycerophosphoinositol in any of the tissues. The most prominent change concerned inositol 1,4-bisphosphate, which increased enormously in the insect salivary gland and parotid gland after stimulation with 5-hydroxytryptamine and carbachol respectively. Carbachol also induced a large increase in the level of inositol 1,4,5-trisphosphate in the parotid. Stimulation of brain slices with carbachol induced modest increase in the bis- and tris-phosphate. In all the tissues studied, there was a significant agonist-dependent increase in the level of inositol 1-phosphate. The latter may be derived from inositol 1,4-bisphosphate, because homogenates of the insect salivary gland contain a bisphosphatase in addition to a trisphosphatase. These results suggest that the earliest event in the stimulus-response pathway is the hydrolysis of polyphosphoinositides by a phosphodiesterase to yield inositol 1,4,5-trisphosphate and inositol 1,4-bisphosphate, which are subsequently hydrolysed to inositol 1-phosphate and inositol. The absence of inositol 1:2-cyclic phosphate could indicate that, at very short times after stimulation, phosphatidylinositol is not catabolized by its specific phosphodiesterase, or that any cyclic derivative liberated is rapidly hydrolysed by inositol 1:2-cyclic phosphate 2-phosphohydrolase.     

A novel metal-dye detection system permits picomolar-range h.p.l.c. analysis of inositol polyphosphates from non-radioactively labelled cell or tissue specimens.

A novel complexometric dye- and transition-metal-based post-column detection system for polyanions, called 'metal-dye detection' has been developed. This technique, combined with a new h.p.l.c. separation protocol, permits a direct highly-isomer-selective determination of bis- to poly-phosphorylated non-radioactively labelled compounds in the picomolar range, a sensitivity hitherto unknown for these substances. The application of the technique in the quantitative microanalysis of inositol polyphosphates from milligram amounts of cells or tissue specimens is described. The technique promises to answer hitherto unresolved questions about the role of inositol phosphates, especially those in intact tissues, which are not readily amenable to analysis by radioisotopic techniques.     

Orthovanadate and fluoroaluminate stimulate inositol phosphate production and in vitro ovulation in goldfish (Carassius auratus) follicles

Both sodium orthovanadate and fluoroaluminate were found to stimulate in vitro ovulation in intact goldfish follicles, suggesting the involvement of G-proteins in ovulation. Although orthovanadate was able to stimulate cAMP production, it probably stimulates ovulation by some other mechanism since cAMP blocks ovulation in this species. These agents also stimulated the accumulation of labeled inositol phosphate in follicle walls. The time course of inositol phosphate production showed a slightly delayed and continuous accumulation for isomers of inositol mono-, bis-, and trisphosphates. No change was observed in inositol tetrakisphosphate levels over time. The accumulation of inositol phosphates in response to orthovanadate was also dose-dependent. Lithium chloride (10 mM) caused varying increases in the levels of most isomers and a decrease in ins-3,4-P2. Inositol phosphate production varied significantly with changes in the maturational stage of follicles. Peak production was observed in follicles 7-8 h after hCG treatment, which corresponds almost exactly with the time of ovulation. This correlation of maximal inositol phosphate production with the time of ovulation, along with the stimulation of ovulation by diacylglycerols, a phorbol ester, and the G-protein-stimulating agents, orthovanadate and fluoroaluminate, suggests a role for polyphosphatidylinositol hydrolysis in ovulation.     

Phospholipase C activation induced by noradrenaline in rat hippocampal slices is potentiated by GABA-receptor stimulation.

We have studied the effect of gamma-aminobutyric acid (GABA) and other GABA-receptor agonists (3-aminopropanesulphonic acid and muscimol) on the noradrenaline-induced stimulation of polyphosphoinositide metabolism in rat hippocampal slices. Formation of water-soluble inositol phosphates, and polyphosphoinositide metabolism were studied in hippocampal slices prelabelled with [3H]myoinositol. Noradrenaline induced formation of inositol mono-, bis- and trisphosphate during 10 min incubation in the presence of lithium; activation of phospholipase C by noradrenaline was also reflected by the hydrolysis of polyphosphoinositides and by the increased metabolism of phosphatidylinositol. GABA-receptor agonists were unable to activate per se phospholipase C; however, when added together with a low concentration of noradrenaline, they greatly potentiated the noradrenaline-stimulated polyphosphoinositide metabolism. We conclude that GABA-receptor agonists potentiate the effect of noradrenaline on polyphosphoinositide turnover and we discuss the role of this neurotransmitter interaction in the physiology of the hippocampus.     

Phytase from Wheat Bran

myo-Inositol mono-, di-, tri-, tetra-, and pentaphosphate were prepared by enzymic hydrolysis of myo-inositol hexaphosphate with a 1,500-fold purified phytase preparation from wheat bran and the subsequent Dowex 1 column chromatography. Relative initial rates of hydrolysis of these inositol phosphates by phytase were nearly the same each other and the activation energy of hydrolysis was about 11,000 cal. per mole for all these substrates. Km values did not vary widely with the substrates. The hydrolysis of inositol phosphates proceeded in a complicated way, except inositol monophosphate, where the reaction was of the first order. The enzyme hydrolyzed the substrates in the manner that removed phosphate group of them one by one. When mixed substrate was used the enzyme showed a preferential attack on the highest member of the phosphates present. From the mixed substrate test, it was concluded that wheat bran phytase is a single enzyme.     

Thrombin-induced phosphoinositide hydrolysis in platelets. Receptor occupancy and desensitization.

The relationship between occupancy of thrombin receptors on platelets and enhanced phosphoinositide hydrolysis was analysed by examination of the dose-response relationship, the effects of thrombin inhibitors and the contribution of secondary effects. Washed human platelets were labelled with [3H]inositol, and agonist-induced accumulation of labelled inositol phosphates was measured. The dose-response curves and the time courses for alpha-thrombin- or gamma-thrombin-induced accumulation of inositol phosphates were similar to those for dense-granule secretion. Addition of the thrombin inhibitor hirudin to thrombin-activated platelets revealed that the continuous presence of active thrombin was required to maintain the accumulation of labelled inositol phosphates; the total production of inositol phosphates increased with longer periods of exposure to thrombin, reaching a maximum between 5 and 10 min. After activation with thrombin, the ability of a second, greater, addition of thrombin to induce additional phosphoinositide hydrolysis decreased with time; it was absent within 10 min after the first addition. The failure to sustain accumulation of labelled inositol phosphates or to respond to a second addition of thrombin beyond 10 min was not due to depletion of the pool of labelled precursors, because the platelets retained their ability to respond to collagen. Addition of ADP-consuming enzymes decreased sensitivity to thrombin, but inhibition of cyclo-oxygenase with indomethacin did not impair the thrombin-induced hydrolysis of phosphoinositides. It was concluded that thrombin-induced hydrolysis of phosphoinositides has characteristics consistent with mediation by a receptor that is similar to that that triggers dense-granule secretion, requires continuous presence of active thrombin to be maintained, is mediated by a receptor that displays thrombin-induced desensitization, and is only partially enhanced by secondary agents.     

Multiple isomers of phosphatidyl inositol monophosphate and inositol bis- and trisphosphates from filamentous fungi

Abstract The range of inositol phosphates and inositol phospholipids present in three filamentous fungi, Neurospora crassa, Fusarium graminearum and Phanerochaete chrysosporium has been investigated by HPLC analysis. The profiles obtained demonstrate that two isomers of phosphatidyl inositol monophosphate are present, and that an apparent complexity in the number of isomers of inositol bis- and trisphosphates is found in filamentous fungi that has not been observed in animal or plant cells.     

Activation of polyphosphoinositide phospholipase C by guanosine 5'-O-(3-thio)triphosphate and fluoroaluminate in membranes prepared from a human T cell leukemia line, JURKAT

Polyphosphoinositide hydrolysis was studied in membranes prepared from a human T cell leukemia line, JURKAT, prelabeled with myo-[2-3H]inositol. The formation of inositol bis- and trisphosphates was stimulated in a buffer with 110 nM free Ca2+ with a nonhydrolyzable GTP analogue, GTP gamma S, and NaF plus AlCl3 in a time- and concentration-dependent manner. GTP gamma S and NaF-AlCl3 had no significant effect on the inositol monophosphate level. AlCl3 enhanced the NaF-stimulated release of inositol polyphosphates. Optimum concentrations of NaF and AlCl3 produced 1.5-fold more inositol polyphosphates than that produced by optimum concentration of GTP gamma S. OKT3 monoclonal antibody, an antibody against the T-cell receptor complex, did not stimulate the inositol polyphosphate formation by JURKAT membranes even in the presence of GTP, although the antibody at the concentrations used markedly stimulated the hydrolysis of polyphosphoinositides in intact JURKAT cells.     

Accumulation of Inositol Phosphates Induced by Chlorpromazine in C6 Glioma Cells

Abstract: Chlorpromazine, a cationic amphiphilic drug known to affect phospholipid metabolism, greatly increases the generation of inositol phosphates in C6 glioma cells. When a pulse-chase protocol with myo-[2-³H]inositol as the radioactive precursor was used, the peak increase in radioactivity of inositol phosphates was observed at 20 min. The drug decreased inositol tetrakisphosphate labeling as a percentage of inositol trisphosphate in a dose-dependent manner. It also increased the labeling of the inositol-containing phospholipids, the precursors of the inositol phosphates. The increase in radioactivity of both phospholipids and inositol phosphates was dose-dependent, but appeared also to be a function of the time of exposure of the cultures to the drug, suggesting that the concentration of chlorpromazine in the cell, and not that in the medium, is the critical factor. The optimum concentration for maximum phospholipid labeling was lower than that eliciting maximum generation of inositol phosphates. The data suggest that the mechanism probably does not involve cell-surface receptors, but rather may consist of a direct effect of chlorpromazine on phosphoinositidase C and possibly other enzymatic reactions concerned with the metabolism of inositol phosphates.     

The labelling of polyphosphoinositides with [32P]Pi and the accumulation of inositol phosphates in vasopressin-stimulated hepatocytes.

When hepatocytes were incubated with [32P]Pi, the kinetics for the labelling of the monoester phosphate groups of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate were similar to each other and slightly slower than that for the labelling of the gamma-phosphate of ATP. Analysis of the water-soluble 3H-labelled materials derived from [3H]inositol-labelled hepatocytes revealed that, in addition to inositol and its mono-, bis- and tris-phosphates (Ins, InsP, InsP2 and InsP3), these cells contained two unidentified radioactive compounds which co-eluted with InsP on anion-exchange chromatography. When [3H]inositol-labelled hepatocytes were stimulated with 0.23 microM-vasopressin in the presence of 10 mM-Li+, there was an accumulation of radioactivity in InsP, InsP2 and InsP3 but not in Ins or the two unidentified compounds. Further analysis of these inositol phosphates by h.p.l.c. revealed that vasopressin also stimulates the accumulation of inositol tetrakisphosphate (InsP4) in these cells. Vasopressin-stimulated InsP and InsP2 accumulations were maximal in the presence of 1-10 mM-Li+ but InsP3 accumulation continued to increase up to 50 mM-Li+. Accumulated inositol phosphates were retained within the cell. Li+ from 1 to 50 mM did not influence the extent of vasopressin-stimulated inositol lipid degradation in hepatocytes. In the absence of Li+, radioactivity in vasopressin-stimulated hepatocytes accumulated almost entirely in free inositol. The vasopressin-stimulated accumulation of inositol phosphates in the presence of 10 mM-Li+ was abolished by a V1-vasopressin antagonist. Inositol phosphate accumulation was not influenced by ionophore A23187, dimethyl sulphoxide or indomethacin.     

Decapitation-induced changes in inositol phosphates in rat brain

Decapitation resulted in a time-dependent production of inositol phosphates in rat brain. This production was analyzed by measuring both the radioactivity and the concentrations of inositol phosphates generated from [3H]inositol-labeled phospholipids. Both measurements produced the same time-dependent changes, including a rapid decrease in inositol 1,4,5-trisphosphate within 1.5 min, a 6-fold increase in inositol 1,4-bisphosphate to a maximum at 1.5 min, a 5-fold rise in inositol 4-monophosphate to a maximum at 2.5 min, and little change in inositol 1-monophosphate. The temporal changes in the mass and radioactivity of these compounds, together with the decrease in labeling of phosphatidylinositol 4,5-bisphosphates, support the idea that the inositol phosphates originate from the hydrolysis of phosphatidylinositol 4,5-bisphosphates and not from either the direct hydrolysis of phosphatidylinositol 4-phosphates or phosphatidylinositols.